Diabetes mellitus is the third leading cause of death in the U.S. and the leading cause of blindness, renal failure, and amputation. Diabetes is also a major cause of premature heart attacks and stroke and accounts for 15% of U.S. health care costs. Approximately 5% of Americans, and as many as 20% of those over the age of 65, have diabetes.
Diabetes results from the failure of the .beta.-cells in the islets of Langerhans in the endocrine pancreas to produce adequate insulin to meet metabolic needs. Diabetes is categorized into two clinical forms: Type 1 diabetes (or insulin-dependent diabetes) and Type 2 diabetes (or non-insulin-dependent diabetes). Type 1 diabetes is caused by the loss of the insulin-producing .beta.-cells. Type 2 diabetes is a more strongly genetic disease than Type 1 (Zonana & Rimoin, 1976 N. Engl. J. Med. 295:603), usually has its onset alter in life, and accounts for approximately 90% of diabetes in the U.S. Affected individuals usually have both a decrease in the capacity of the pancreas to produce insulin and a defect in the ability to utilize the insulin (insulin resistance). Obesity causes insulin resistance, and approximately 80% of individuals with Type 2 diabetes are clinically obese (greater than 20% above ideal body weight). Unfortunately, about one-half of the people in the U.S. affected by Type 2 diabetes are unaware that they have the disease. Clinical symptoms associated with Type 2 diabetes may not become obvious until late in the disease, and the early signs are often misdiagnosed, causing a delay in treatment and increased complications. While the role of genetics in the etiology of type 2 diabetes is clear, the precise genes involved are largely unknown.
Depression and obesity can each be associated with a defect in serotonin production, serotonin metabolism, or serotonin-mediated neurotransmission. Serotonin (5-hydroxytryptamine (5HT)) is a biogenic amine that not only functions as a neurotransmitter (Takaki, et al., 1985 J. Neurosciences 5:1769) and as a hormone, (Kravitz, et al., 1980 J. Exp. Biol. 89:159), but also as a mitogen (Nemeck, et al., 1986 Proc. Natl. Acad. Sci. USA 83:674). In its functions as a neurotransmitter, serotonin modulates many forms of synaptic transmission and is believed to exert a number of effects on neuronal growth during early development. In addition, serotonin is also believed to modulate numerous sensory, motor, and behavioral processes in the mammalian nervous system (see Jacobs. in Hallucinogens: Neurochemical, Behavior, and Clinical Perspectives (eds. Jacobs) 183-202 (Raven, N.Y., 1984), Sleight et al., in Serotonin Receptor Subtypes: Basic and clinical aspects. (eds. Peroutka) 211-227 (Wiley-Liss, New York, N.Y., 1991. Wilkinson et al. in Serotonin receptor subtypes: Basic and clinical aspects (eds. Peroutka) 147-210 (Wily-Liss, New York, N.Y., 1991)). In the cortex, transmission at serotoneurgic synapses contributes to affective and perceptual states; these synapses represent a major site of action of psychotropic drugs such as LSD, Jacobs in Hallucinogens: Neurochemical, Behavioral, and Clinical Perspectives, Jacobs, Ed. (Raven Press, New York, 1984), pages. 183-202.
The diverse responses elicited by serotonin are mediated through the activation of a large family of receptor subtypes, Tecott et al. 1993 Curr. Opin. Neurobiol 3,310-315. The complexity of this signaling system and the paucity of selective drugs have made it difficult to understand development of serotonin-producing cells and to understand the role of serotonin in depression and obesity.
Attempts to understand depression and other serotonin-related disorders have focused upon understanding the development of the brain. Development of the vertebrate forebrain is an elaborate process that gives rise to a variety of essential structures including the cerebral cortex, basal ganglia, hypothalamus and thalamus. Although much is known about these structures and the functions that they perform, very little is understood about the mechanisms that direct their specification, morphogenesis and differentiation. Recently, however, families of candidate regulatory genes with regionally restricted expression in the neuroepithelium of the forebrain have been identified; these gene families are hypothesized to establish positional identity and to control region-specific morphogenesis and histogenesis of the forebrain (Shimamura et al. 1995 Development, 121:3923-3933; Rubenstein, et al., 1994 Science 266:578-581).
Nkx-2.1 and Nkx-2.2 are two of the earliest known genes to be expressed in the neuroectodermal cells of the forebrain; they are expressed at the onset of neurulation in restricted ventral forebrain domains (Shimamura et al., 1995 supra). In addition, expression of these genes is induced by the secreted molecule sonic hedgehog (Shh), a known axial mesendodermal signaling protein that is responsible for the induction of the ventral neurons of the forebrain (Ericson et al., 1995 Cell 81:747-756; Barth and Wilson, 1995, Development 121:1755-1768). The early and spatially limited expression of Nkx-2.1 and Nkx-2.2 in response to a primary inductive signal suggests that these molecules provide the initial positional information for specific ventral regions of the developing forebrain.
Nkx-2.2 is a member of a vertebrate gene family that is homologous to the Drosophila NK-2 gene, which is expressed in neuroblast precursors in the Drosophila head (Kim and Nirenberg, 1989 Proc. Natl. Acad. Sci. USA. 86:7716-7720). The NK-2 gene family is characterized by two regions of homology: the homeobox and a highly conserved sequence downstream of the homeobox.
In addition to its expression in the brain, Nkx-2.2 is also expressed in the pancreas, pancreatic islet .beta. cells, and hamster insulinoma (HIT) cells (Rudnick et al. 1994 Proc. Natl. Acad. Sci. USA 91:12203-12207). Nkx-2.2 expression in pancreas is accompanied by expression of Nkx-6.1, another NK-2-related gene (Rudnick et al. supra). A partial sequence (Price et al. 1992 Neuron 8:241-255) and a full-length sequence of murine Nkx-2.2 (Hartigan and Rubenstein 1996 Gene 168:271-2) have been published. The actual function of Nkx-2.2 or Nkx-6.1 as either transcription factors or as developmental regulators was not previously known.
The pathogenesis of diabetes, depression, and obesity, and the links between these diseases, are complex and not well understood. Moreover, the complex nature of these disorders makes their study difficult. Thus, there is a need for an in vivo model for insulin- and serotonin-producing cells for identification of new compounds for treatment of disorders associated with insulin and serotonin production, as well as for development of new therapies to address such disorders (e.g., methods for replacing or enhancing serotonin-producing and/or insulin-producing cells). In addition, there is a need for a method to identify individuals at risk of developing insulin and serotonin production-associated disorders. Finally, there is little known about the development and differentiation of the pancreatic islet cells or key cell types in the central nervous system.